Bacterial, viral, parasitic and mycoplasma infections are wide spread in the veterinary animals such as cattle, swine and companion animal. Diseases caused by these infectious agents are often resistant to antimicrobial pharmaceutical therapy, leaving no effective means of treatment. Consequently, a vaccinology approach is increasingly used to control the infectious disease in the veterinary animals. A whole infectious pathogen can be made suitable for use in a vaccine formulation after chemical inactivation or appropriate genetic manipulation. Alternatively, a protein subunit of the pathogen can be expressed in a recombinant expression system and purified for use in a vaccine formulation.
Adjuvant generally refers to any material that increases the humoral and/or cellular immune response to an antigen. The traditional vaccines are composed of crude preparation of killed pathogenic microorganisms, and the impurities associated with the cultures of pathological microorganisms could act as adjuvant to enhance the immune response. However, when homogeneous preparations of pathological microorganisms or purified protein subunits are used as antigens for vaccination, the immunity invoked by such antigens is poor and the addition of certain exogenous materials as adjuvant therefore becomes necessary. Further, synthetic and subunit vaccines are expensive to produce. Therefore, with the aid of adjuvant, a smaller dose of antigen may be required to stimulate the immune response, thereby saving the production cost of vaccines.
Adjuvants are known to act in a number of different ways to enhance the immune response. Many adjuvants modify the cytokine network associated with immune response. These immunomodulatory adjuvants can exert their effect even when they are not together with antigens. In general the immunomodulatory adjuvants cause a general up-regulation of certain cytokines and a concomitant down regulation of others leading to a cellular Th1 and/or a humoral Th2 response.
Some adjuvants have the ability to preserve the conformational integrity of an antigen so that the antigens can be efficiently presented to appropriate immune effector cells. As a result of this preservation of antigen conformation by the adjuvant formulation, the vaccine would have an increased shelf-life such as that shown for immune stimulating complexes (ISCOMs). Ozel M., et. al.; Quarternary Structure of the Immunestimmulating Complex (Iscom), J. of Ultrastruc. and Molec. Struc. Res. 102, 240-248 (1989).
Some adjuvants have the property of retaining the antigen as a depot at the site of injection. As a result of this depot effect the antigen is not quickly lost by liver clearance. Aluminum salts and the water-in-oil emulsions act through this depot effect for a shorter duration. For example, one can obtain a long-term depot by using Freund's complete adjuvant (FCA) which is an water-in-oil emulsion. FCA typically remains at the injection site until biodegradation permits removal of the antigen by antigen-presenting cells.
Based on their physical nature, adjuvants can be grouped under two very broad categories, namely particulate adjuvants and non-particulate adjuvants. Particulate adjuvants exist as microparticles. The immunogen is either able to incorporate or associate with the microparticles. Aluminum salts, water-in-oil emulsions, oil-in-water emulsions, immune stimulating complexes, liposomes, and nano- and microparticles are examples of particulate adjuvants. The non-particulate adjuvants are generally immunomodulators and they are generally used in conjunction with particulate adjuvants. Muramyl dipeptide (an adjuvant-active component of a peptidoglycan extracted from Mycobacteria), non-ionic block copolymers, Saponins (a complex mixture of triterpenoids extracted from the bark of the Quillaja saponaria tree), Lipid A (a disaccharide of glucosamine with two phosphate groups and five or six fatty acid chains generally C12 to C16 in length), cytokines, carbohydrate polymers, derivatized polysaccharides, and bacterial toxins such as cholera toxin and E. coli labile toxin (LT) are examples of non-particulate adjuvants.
Some of the best-known adjuvants are combination of non-particulate immunomodulators and particulate materials which could impart depot effect to the adjuvant formulation. For example, FCA combines the immunomodulatory properties of Mycobacterium tuberculosis components along with the short-term depot effect of oil emulsions.
Oil emulsions have been used as vaccine adjuvant for a long time. Le Moignic and Pinoy found in 1916 that a suspension of killed Salmonella typhimurium in mineral oil increased the immune response. Subsequently in 1925, Ramon described starch oil as one of the substances augmenting the antitoxic response to diptheria toxoid. However, the oil emulsions did not become popular until 1937 when Freund came out with his adjuvant formulation now known as Freund's Complete Adjuvant (FCA). FCA is a water-in-oil emulsion composed of mineral (paraffin) oil mixed with killed Mycobateria and Arlacel A. Arlacel A is principally mannide monooleate and is used as an emulsifying agent. Although FCA is excellent in inducing an antibody response, it causes severe pain, abscess formation, fever and granulomatous inflammation. To avoid these undesirable side reactions, Incomplete Freund's Adjuvant (IFA) was developed. IFA is similar to FCA in its composition except for the absence of mycobacterial components. IFA acts through depot formulation at the site of injection and slow release of the antigen with stimulation of antibody-producing cells.
Another approach to improve FCA was based on the notion that replacing the mineral oil with a biocompatible oil would help eliminate the reactions associated with FCA at the injection site. It was also believed that the emulsion should be oil-in-water rather than water-in-oil, because the latter produces a long-lasting depot at the injection site. Hilleman et al. described an oil-based adjuvant “Adjuvant 65”, consisting of 86% peanut oil, 10% Arlacel A as emulsifier and 4% aluminum monostearate as stabilizer. Hilleman, 1966, Prog. Med. Virol. 8: 131-182; Hilleman and Beale, 1983, in New Approaches to Vaccine Development (Eds. Bell, R. and Torrigiani, G.), Schwabe, Basel. In humans, Adjuvant 65 was safe and potent but exhibited less adjuvanticity than IFA. Nevertheless, the use of Adjuvant 65 was discontinued due to reactogenicity for man with certain lots of vaccine and reduction in adjuvanticity when a purified or synthetic emulsifier was used in place of Arlacel A. U.S. Pat. Nos. 5,718,904 and 5,690,942 teach that the mineral oil in the oil-in-water emulsion can be replaced with metabolizable oil for the purpose of improving the safety profile.
Besides the adjuvanticity and safety, the physical appearance of an emulsion is also an important commercial consideration. Physical appearance depends on the stability of the emulsion. Creaming, sedimentation and coalescence are indicators of the emulsion instability. Creaming occurs when oil and aqueous phases of the emulsion have different specific gravity. Creaming also occurs when the initial droplet size of the emulsion is large and the emulsion droplets are not having any Brownian motion. When the droplet size is large, there is a tendency for the interfacial rupture and the droplets coalesce into large particles. The stability of the emulsion is determined by a number of factors such as the nature and amount of emulsifier used, the size of the droplet size in the emulsion, and the difference in the density between the oil and water phase.
Emulsifiers promote stabilization of dispersed droplet by reducing the interfacial free energy and creating physical or electrostatic barriers to droplet coalescence. Nonionic as well as ionic detergents have been used as emulsifiers. Nonionic emulsifiers orient at the interface and produce relatively bulky structures, which leads to steric avoidance of the dispersed droplets. Anionic or cationic emulsifiers induce formation of an electrical double layer by attracting counter ions; the double layer repulsive forces cause droplets to repel one another when they approach.
Besides using the emulsifiers, the stability of the emulsion can also be achieved through reducing the droplet size of the emulsion by mechanical means. Typically propeller mixers, turbine rotors, colloid mills, homogenizers, and sonicators have been used to manufacture emulsions. Microfluidization is another way to increase the homogeneity of the droplet size in the emulsion. Microfluidization can produce an elegant, physically stable emulsion with consistent particle size in the submicron range. Besides increasing the stability of the emulsion, the process of microfluidization allows terminal filtration which is a preferred way of ensuring the sterility of the final product. Moreover, submicron oil particles can pass from injection sites into the lymphatics and then to lymph nodes of the drainage chain, blood and spleen. This reduces the likelihood of establishing an oily depot at the injection site which may produce local inflammation and significant injection site reaction.
Microfluidizers are now commercially available. Emulsion formation occurs in a microfluidizer as two fluidized streams interact at high velocities within an interaction chamber. The microfluidizer is air or nitrogen driven and can operate at internal pressures in the excess of 20,000 psi. U.S. Pat. No. 4,908,154 teaches the use of microfluidizer for obtaining emulsions essentially free of any emulsifying agents.
A number of submicron oil-in-water adjuvant formulations have been described in the literature. U.S. Pat. No. 5,376,369 teaches a submicron oil-in-water emulsion adjuvant formulation known as Syntax Adjuvant Formulation (SAF). SAF contains squalene or squalane as the oil component, an emulsion-forming amount of Pluronic L121 (polyoxy-propylene-polyoxyethylene) block polymer and an immunopotentiating amount of muramyldipeptide. Squalene is a linear hydrocarbon precursor of cholesterol found in many tissues, notably in the livers of sharks and other fishes. Squalane is prepared by hydrogenation of squalene and is fully saturated. Both squalene and squalane can be metabolized and have a good record of toxicological studies. Squalene or squalane emulsions have been used in human cancer vaccines with mild side effects and a desirable efficacy. See, e.g., Anthony C. Allison, 1999, Squalene and Squalane emulsions as adjuvants, Methods 19:87-93.
U.S. Pat. No. 6,299,884 and International Patent Publication WO 90/14837 teach that the polyoxy-propylene-polyoxyethylene block copolymers are not essential for the formation of submicron oil-in-water emulsion. Moreover, these references teach the use of non-toxic, metabolizable oil and expressly exclude the use of mineral oil and toxic petroleum distillate oils in their emulsion formulations.
U.S. Pat. No. 5,961,970 teaches yet another submicron oil-in-water emulsion to be used as a vaccine adjuvant. In the emulsion described in this patent, the hydrophobic component is selected from the group consisting of a medium chain triglyceride oil, a vegetable oil and a mixture thereof. The surfactant included in this emulsion can be a natural biologically compatible surfactant such as phospholipid (e.g., lecithin) or a pharmaceutically acceptable non-natural surfactant such as TWEEN-80. This patent also teaches incorporating the antigen into the emulsion at the time the emulsion is formed, in contrast to mixing the antigen with the emulsion after the emulsion has been independently and extrinsically formed.
U.S. Pat. No. 5,084,269 teaches that an adjuvant formulation containing lecithin in combination with mineral oil causes a decrease in irritation within the host animal and simultaneously induces increased systemic immunity. The adjuvant formulation resulting from U.S. Pat. No. 5,084,269 is commercially used in veterinary vaccines under the trade name AMPHIGEN®. The AMPHIGEN® formulation is made up of micelles—oil droplets surrounded by lecithin. These micelles allow more whole cell antigens to attach than traditional oil-based adjuvants. Moreover, the AMPHIGEN®-based vaccine formulations contain a low oil content of 2.5 to 5% mineral oil, compared to other vaccine formulations containing oil adjuvants, which typically contain from 10% to 20% oil. Its low oil content makes this adjuvant-based vaccine formulation less irritating to tissues at the injection site, resulting in fewer lesions and less trim at slaughter. In addition, the lecithin coating surrounding the oil droplets further reduces injection site reactions resulting in a vaccine that is both safe and efficacious.
The AMPHIGEN® formulation is used as an adjuvant in a number of veterinary vaccines and there is need to maintain the physical appearance of the vaccine product during short and long storage periods as well as at the time of reconstitution. In addition, a lyophilized antigen is mixed with the pre-made adjuvant formulation just before the injection. This practice does not always ensure that there is a uniform distribution of the antigen within the oil-in-water emulsion and the appearance of the emulsion may not be desirable. Moreover, upon standing, the homogenized emulsion can show phase separation. Therefore, there exists a need for a stable adjuvant formulation which does not show phase separation upon long shelf-life. One way to prevent the phase separation is to reduce the droplet size and increase the particle homogeneity of the emulsion. While the process of microfluidization of metabolizable oil-based emulsion formulations has been documented, microfluidization of oil-in-water emulsions such as the AMPHIGEN® formulation has not yet been carried out.
In the present invention, microfluidization has been used to bring the size of lecithin-surrounded mineral oil droplets to submicron size. Unexpectedly, it has been discovered by the present inventors that microfluidization of vaccine formulations adjuvanted with an oil-in-water emulsion comprised of a mixture of lecithin and oil not only improves the physical appearance of the formulations, but also enhances the immunizing effects of the formulations. Microfluidized formulations are also characterized by an improved safety profile.